RESEARCH NOTE J our nal of Nemat ol ogy 27(2):244-247. 1995.

The Society of Nematologists 1995.

A Rapi d and Si mpl e Met hod f or Stai ni ng Lipid in
Fi xed Nemat odes 1
WM. TERRELL STAMPS AND MARC J . LI NI T 2
Abstract: A met hod is descri bed f or staining lipid in fourt h-st age dispersal j uveni l e nemat odes
fi xed with formal-acetic fixative (FA4:I). Bursaphelenchus xylophilus fourt h-st age dispersal j uveni l es
were fi xed with hot FA4:1 f or 24 hours, excess fixative was r emoved, and a solution of sat urat ed oil
r ed O in 96% et hanol added and allowed to sit f or 25 minutes at 60 C. Excess oil r ed O was r emoved,
nemat odes wer e washed twice with 70% ethanol, and were processed to pur e glycerin. Li pi d dropl et s
within t he nemat odes were viewed by light microscopy and appear ed as dark r ed spheres of vari ous
sizes. Comput er i zed i mage analysis was used to quant i fy lipid dr opl et area.
Key words: Bursaphelenchm xflophilus, pi ne wilt, pi newood nemat ode, oil r ed O, lipid, staining.
Li pi d is essential f or nemat ode survival
and is t he maj or sour ce of ener gy f or t he
dauer l ar vae or infective stages of virtually
all ani mal and plant-parasitic nemat odes
(1,3). The fat e of lipid unde r vari ous con-
ditions and t he rat e o f its utilization are of
i mpor t ance in unde r s t a ndi ng ne ma t ode
ecology, behavi or, and physiology. Ener gy
ut i l i zat i on, met abol i c r at es, a nd agi ng
pr ogr ess can be det er mi ned f r om t he lipid
cont ent of nemat odes (1,10).
Bursaphelenchus xylophilus (St ei ner and
Buhr er , 1934) Nickle, 1970 is t he causal
agent of pi ne wilt (4). The f our t h- st age dis-
per sal j uveni l e o f B. xylophilus is a nonf eed-
i ng st age t hat is morphol ogi cal l y and phys-
iologically distinct f r om ot her life stages. It
cont ai ns a l arge amount of lipid t hat is uti-
lized as an ener gy sour ce (5,6). Four t h-
stage di spersal j uveni l es ar e car r i ed f r om
i nf ect ed pi nes to new hosts by cerambyci d
beet l es, Monochamus spp. (7). We ar e in-
t er est ed in t he rel at i onshi p bet ween lipid
cont ent and exit behavi or of dispersal j u-
veniles f r om beet l e vectors. Thi s paper re-
por t s on a lipid-staining t echni que (modi-
fication of Croll (2) and Sei nhorst (8)) t hat
Received for publication 19 September 1994.
1 Contribution from the Missouri Agricultural Experiment
Station. Journal Series No. 12,258.
Department of Entomology, University of Missouri, Co-
lumbia, MO 65211. Address all correspondence to M. J.
Linit.
244
facilitates quant i fi cat i on of lipid cont ent in
single or a small numbe r of nemat odes.
The t echni que was modi f i ed f r om t he
wor k of Croll (2). I n Croll' s st udy, t he non-
f eedi ng infective stage of t he hook wor m
Ancylostoma tubaeforme (Zeder), a ver t ebr at e
parasite, was alive in wat er when st ai ned
with oil r ed O and pr ocessed to glycerin by
a met hod modi f i ed f r om Sei nhor st (8).
The nemat odes wer e t hen anal yzed with a
scanni ng mi cr odens i t omet er at 517 nm.
Our met hod is f or fi xed speci mens.
MATERIALS AND METHODS
Four t h- s t age di sper sal j uveni l es wer e
obt ai ned f r om newly emer ged adul t Mono-
chamus carolinensis (Olivier) beet l es t hat had
devel oped in j ack pi ne, Pi nus banksiana
Lamb. , l ogs i nf ect ed wi t h B. xylophilus.
Nemat odes were collected f r om adul t bee-
tles using a modi f i ed Baer mann t echni que
(9) and wer e t r ansf er r ed to wells of a 24-
well plastic t i ssue cul t ur e pl at e (Fal con
#3047, Bect on Di ckenson, Li ncol n Park,
N J). Most of t he wat er was r emoved f r om
each well with a Past eur pi pet t e unde r a
dissecting mi croscope to pr event r emoval
o f n e ma t o d e s . Fol l owi ng a s t a n d a r d
met hod for fixing nemat odes, 2- 3 ml of
hot (90 C) f or mal - acet i c fixative 4:1 (FA4:
1; 10 parts formal i n (40% f or mal dehyde) ,
1 par t glacial acetic acid, 89 part s distilled
water) was quickly added t o each well (9).
The tissue cul t ur e plate was covered, kept
Staining Lipid in Fixed Nematodes: Stamps, Linit 245
at room t emperat ure for 24 hours, and
t hen most of t he fixative was removed
from each well with a Pasteur pipette un-
der magnification, as described above.
For our staining method, a saturated so-
lution of oil red O (no. 0-0625, Sigma
Chemical Co., St. Louis, MO) in ethanol
was made by addi ng 1-3 g of oil red O
powder to 100 ml of 96% ethanol (Croll (2)
used 70% ethanol) and was stirred for 20
minutes. The solution was t hen filtered
t hrough a vacuum filter apparatus using a
What man no. 2 paper filter.
Each well received 1.5 ml of the satu-
rated oil red O solution, and the plate was
covered and held at 60 C for 25 minutes in
an oven (2). Upon removal from the oven,
most of the oil red O solution was removed
from each well with a Pasteur pipette and 2
ml of 70% ethanol was added to each well.
After sufficient time had elapsed for the
nematodes to settle to the bottom of each
well (about 10 minutes), the excess ethanol
was removed with a Pasteur pipette. The
ethanol wash was repeated a second time
(modified from distilled water washes as
described in Croll (2)).
Instead of addi ng a 50/50 (V:V) water-
glycerin Solution to the wells and allowing
the water to evaporat e as described by
Croll (2), we processed the nematodes to
glycerin in the wells of the tissue culture
plate following a modification of the quick
met hod of Sei nhorst (8,9). Two ml of
"Seinhorst I" solution (20 parts 96% etha-
nol, 1 part glycerin, 79 parts distilled wa-
ter) at room t emperat ure (21 C) was added
to each well. The uncovered tissue culture
plate was placed in a closed vessel contain-
ing a small volume of 96% ethanol. This
vessel was placed in an oven at 35-40 C for
24 hours. The excess liquid in each well
was removed with a Pasteur pipette, and 2
ml of a modified "Seinhorst II" solution
(10 parts glycerin, 90 parts 96% ethanol) at
room t emperat ure (21 C) was added. The
uncovered tissue culture plate, not in the
closed vessel, was ret urned to the oven un-
til all ethanol had evaporated, by which
time the stained nematodes were t hen in
pure glycerin and ready to mount. The
nematodes were mount ed in drops of pure
glycerin on standard microscope slides us-
ing the wax ring met hod (9) and viewed
with the use of transmitted light micros-
copy.
RESULTS AND DISCUSSION
Once the nematodes were in pure glyc-
erin, the lipid droplets appeared as dark
red spheres of various sizes and were quite
obvi ous whe n t he ne ma t ode s wer e
mount ed and viewed under light micros-
copy (Fig. 1). The lipid droplets within the
nematodes did not appear to be red or ap-
peared to be stained very lightly red while
going t hrough the process. This was usu-
ally an artifact of processing.
Chemical methods of quantifying lipid
content oft en require large numbers of
specimens, and they are impractical for
single or a few nematodes. The met hod
presented here is excellent for examining
lipid in a very small number of specimens
because individuals can be tracked under a
microscope t hroughout the process. Use of
a 24-well tissue culture plate also allows a
large number of specimens or groups of
specimens to be processed at one time and,
at the end of the processing, provides con-
venient long-term storage. Specimen in-
formation can be recorded on the lid. This
technique produces preserved specimens
that can be re-examined later. Nematodes
already in glycerin appear unhar med after
going t hrough part of the process a sec-
ond, third, or fourt h time.
The intensity and precision of the stain-
ing for lipids have allowed us to quantify
lipid content in individual nematodes. Im-
ages of nematodes under transmitted light
microscopy were recorded by a video cam-
era attached to a video capture board in
a personal comput er and analyzed with
MOCHA (Jandel Scientific, San Raphael,
CA), an image analysis program that can
distinguish colors and levels of density.
Lipid droplet area and whole body area
were quantified, and percentage lipid area
(lipid per body) was used as a measure of
lipid content for fourth-stage dispersal ju-
veniles under various t reat ment regimes.
246 Journal of Nematology, Volume 27, No. 2, June 1995
FIG. 1. Photomicrographs of A) an unstained Bursaphelenchus xylophilus fourth-stage dispersal juvenile and
B) an oil red O-stained fourth-stage dispersal juvenile. Red-stained lipid droplets fill much of the body of the
nematode and appear black in the photomicrograph. Head region in A and B at upper left. Scale bar =
0.1 ram.
T h e t e c h n i q u e p r e s e n t e d h e r e wor ks
v e r y wel l f o r s t a i ni ng f o u r t h - s t a g e di s-
per s al j uve ni l e s o f t he n e ma t o d e B. xylo-
philus a n d s houl d wor k equal l y well wi t h
o t h e r f i xed n e ma t o d e speci es t hat cont ai n
l i pi d dr opl et s . T h e me t h o d is conveni ent ,
r api d, a nd cons i s t ent i n t he uni f or mi t y a n d
densi t y o f st ai ni ng pr ovi de d.
Staining Lipid in Fixed Nematodes: Stamps, Linit 247
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